Method and apparatus for determining target depth, brightness and size within a body region

ABSTRACT

A method of investigating the location and size of a light-emitting source in a subject is disclosed. In practicing the method, one first obtains a light intensity profile by measuring, from a first perspective with a photodetector device, photons which (i) originate from the light-emitting source, (ii) travel through turbid biological tissue of the subject, and (iii) are emitted from a first surface region of interest of the subject. The light-intensity profile is matched against with a parameter-based biophotonic function, to estimate function parameters such as depth and size. The parameters so determined are refined using data other than the first measured light intensity profile, to obtain an approximate depth and size of the source in the subject. Also disclosed is an apparatus for carrying out the method.

[0001] This application claims priority to Provisional ApplicationSerial No. 60/291,794 filed on May 17, 2001, which is incorporated inits entirety herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to an apparatus and method for fields ofnon-invasive medical imaging, medical research, pathology, and drugdiscovery and development.

BACKGROUND OF THE INVENTION

[0003] In a variety of medical diagnostic and therapeutic settings, aswell as in biomedical research, it is desirable to image a subsurfacetarget or area within a body region of a subject. For example,non-invasive locating and imaging of part or all of a solid tumor, anarea of myocardial ischemia, the distribution of a therapeutic compoundadministered to the subject, or the progression of a disease may provideuseful research or diagnostic information. Ideally, an imaging method isable to locate within a body region a target of interest, and provideinformation about the target's shape, size, number of cells, and depthbelow the surface of the body region. However, until now, methods thathave been used and/or proposed for subsurface body target imaging havegenerally been limited to those using ionizing radiation such as X-rays,expensive and bulky equipment such as Magnetic Resonance imaging (MRI),or ultra-sound.

[0004] X-rays have excellent tissue penetration, and when used inconjunction with computed tomography (CT) or computed axial tomography(CAT), can produce superior image quality. However, X-rays have limiteduse in monitoring disease progress because exposure to X-rays ispotentially harmful if such exposure is prolonged. X-rays can be used tolocate and image compositions that have localized at a target within abody region, but always with exposure to the potential harm associatedwith X-ray radiation. X-rays, however, cannot readily be used to imagethe expression of gene products in vivo and determine the depth and/orshape of a target expressing such gene products.

[0005] MRI is also an excellent method for imaging targets, areas, andstructures in a body region of a subject. Although MRI is not thought topossess harmful attributes like those associated with ionizingradiation, the expense and bulky equipment size needed to use MRI makeit impractical for many applications or situations. MRI can provide twoand three-dimensional information about targets within a body region ofa subject, but is less effective at imaging physiological activityassociated with a target.

[0006] Ultrasound or ultrasonography is the use of high-frequency sound(ultrasonic) waves to produce images of structures within the humanbody. Ultrasonic waves are sound waves that are above the range of soundaudible to humans. Ultrasonic waves are produced by the electricalstimulation of a piezoelectric crystal and such waves can be aimed atspecific body regions. As the waves travel through body tissues within abody region, they are reflected back at any point where there is achange in tissue density, as, for instance, in the border between twodifferent organs of the body. Ultrasound offers the advantages of notusing radiation or radioactive material, and employs lesser expensiveand less bulky equipment than MRI, but is limited to only discerningdifferences in density of underlying tissue and structures. Accordingly,ultrasound cannot effectively track and monitor the progress of aninfection unless such infection results in a discernable shift indensity of the target tissue. Ultrasound cannot image or detect thephysiological functions of tissues or organs.

[0007] Until now, Positron Emission Tomography or P.E.T. was uniqueamong imaging techniques because it produces an image of organ or tissuefunction. Other imaging techniques such as X-ray, CT, MRI, andsonography depict organ or tissue anatomy but cannot discernphysiological activity within them. To image a specific biochemicalactivity of an organ, a radioactive substance, called a radiotracer orradiopharmaceutical, is injected into the body or inhaled. The tracer isusually a radioactive equivalent of a substance that occurs naturallywithin the body such as water or sugar. The radioactive isotope isidentical to the body's own nonradioactive isotope except that its atomshave a different number of neutrons. Consequently, a subjects body isburdened with radioactive material, and the potential harm associatedwith such material. P.E.T cannot detect non-isotopic expression productsfrom transgenic tissues, organs, or transgenic organisms. Scintigraphy,a diagnostic technique in which a two-dimensional picture of a bodilyradiation source is obtained by the use of radioisotopes, may also beused for imaging structures and their functions. Scintigraphy, however,is not suitable for determining the depth of a target in a body regionof a subject.

[0008] In view of the above-mentioned technologies for locating andimaging a target in a body region of a subject, there is a need formethods and devices to determine the depth and/or the shape and/ornumber of cells of such target without having to use radioactivity,radiation, or expensive and bulky equipment. The invention disclosedherein meets these needs.

SUMMARY OF THE INVENTION

[0009] In one aspect, the invention includes a method of investigatingthe location, size and number of cells, of a light-emitting source in asubject. In practicing the method, one initially obtains a firstmeasured light intensity profile constructed by measuring, from a firstperspective with a photodetector device, photons which (i) originatefrom the light-emitting source, (ii) travel through turbid biologicaltissue of the subject, and (iii) are emitted from a first surface regionof interest of the subject. The light-intensity profile is matchedagainst a parameter-based biophotonic function to estimate functionparameters such as depth and size. The parameters so determined arerefined using data other than the first measured light intensityprofile, to obtain an approximate depth and size of the source in thesubject. The additional data may be data measured from the subject, datafrom modeling analysis, or data relating to the wavelength of photonsemitted from the surface of the subject. As examples:

[0010] The method typically includes generating a 2-D or 3-D visualrepresentation of the light-emitting source, using the approximate depthand shape of the source in the subject, and superimposing the visualrepresentation onto a 2-D or 3-D image of the subject.

[0011] The additional data may be obtained from computer simulation ofthe diffusion of light from a light-emitting source in a turbid medium.One preferred simulation approach involves (i) generating a plurality oftheoretical light-intensity profiles, based on a model of photondiffusion from a glowing source located at one of a plurality of depths,and having one of a plurality of sizes and shapes, through a turbidmedium having absorption and scattering properties similar to those ofbiological tissue, (ii) comparing the quality of fit between each of theplurality of theoretical light-intensity profiles and the first measuredlight intensity profile, (iii) selecting the theoretical light intensityprofile which provides to the first measured light intensity profile,and (iv) obtaining an approximate depth, shape and brightness of thesource in the subject using parameters from the theoretical lightintensity profile selected in (iii). The method may include employing ina photon-scattering model, one or more predetermined tissue-specificlight-scattering coefficients corresponding to tissue through which thephotons travel.

[0012] In another general embodiment, the additional data are obtainedby measuring light emission from the subject at two or more differentwavelengths between about 400 nm and about 1000 nm, determining therelative light intensities measured at the different wavelengths, andcomparing the determined relative light intensities with known relativesignal intensities at the different wavelengths, as a function of tissuedepth. Alternatively, the spectrum of light intensities is measured overa selected wavelength range between about 400-1000 nm, and the measuredspectrum is compared with a plurality of spectra measured fromlight-emitting sources placed at various depths within tissue, todetermine the depth of the light-emitting source from matching themeasured spectrum with the known spectra.

[0013] In various embodiments for a light-emission source, the source isa luminescent moiety or a fluorescent moiety; the light-emitting sourceis administered to the subject and binds to a selected target or isotherwise localized in the subject prior to the measuring; and thelight-emitting source is a light-generating protein (e.g., a luciferase,a green fluorescent protein, etc.), such as a light-generating proteinexpressed by biological cells of the subject or biological cellsadministered to the subject.

[0014] In another aspect, the invention includes apparatus for use ininvestigating the location and size of a light-emitting source in asubject. The apparatus includes a light-tight enclosure within whichlight-emission events from the subject can be detected and an opticalsystem associated with the enclosure for use in generating a first lightintensity profile constructed by measuring, from a first perspectivewithin the enclosure, photons which (i) originate from thelight-emitting source, (ii) travel through turbid biological tissue ofthe subject, and (iii) are emitted from a first surface region ofinterest of the subject. A computational unit operatively connected tothe photo-detector functions to (i) fit the first measured lightintensity profile with a parameter-based biophotonic function; and (ii)refine the parameters of the biophotonic function, using data other thanthe first measured light intensity profile, to generate an approximatedepth and shape of the source in the subject.

[0015] The optical system preferably includes an intensified or cooledcharge-coupled-device (CCD), and a lens for focusing light onto the CCD.The optical system may configured in such a way as to detect photonsemitted from a plurality of different selected surface regions ofinterest of the subject. The system may include one or more filters fortransmitting photons within different selected wavelength ranges, e.g.,above and below 600 nm.

[0016] The computational unit may include a data file of modelbiophotonic functions representing light-emitting sources of varioussizes at various depths, for curve matching with the first spatialprofile.

[0017] Where the optical system includes filters for wavelengthdiscrimination, the computation unit is operable to carry out at leastone of the parameter-refinement operations:

[0018] (i) determining the relative light intensities measured at thedifferent wavelengths, and comparing the determined relative lightintensities with known or calculated relative signal intensities at thedifferent wavelengths, as a function of tissue depth; and

[0019] (ii) comparing the measured spectrum with a plurality of spectrameasured from light-emitting sources placed at various depths withintissue, and determining the depth of the light-emitting source frommatching the measured spectrum with the known spectra.

[0020] In another embodiment, the computational unit is operable toaverage the intensity pattern image into a single intensity value, andfurther to determine source size by integrated light intensity valuesgenerated as a function of a light-emitting source of a particular sizeand shape.

[0021] The computational unit may have a database containing a pluralityof theoretical light-intensity profiles, based on a model of photondiffusion from a light-emitting source located at one of a plurality ofdepths, and having one of a plurality of sizes and shapes, through aturbid medium having absorption and scattering properties similar tothose of biological tissue. Here the computational unit is operable to(i) compare the quality of fit between each of the plurality oftheoretical light-intensity profiles and first measured light intensityprofile, (ii) select the theoretical light intensity profile whichprovides the best fit to the first measured light intensity profile, and(iii) obtain an approximate depth and shape of the source in the subjectusing parameters from the theoretical light intensity profile selectedin (ii).

[0022] In addition, the computational unit is operable to generate avisual 2-, or 3-dimensional representation of the light-emitting source,using the approximate depth and shape of the source in the subject, andsuperimposing the visual representation onto a 2- or 3-dimensional imageof the subject.

[0023] In another aspect, the invention provides a method of determiningthe depth of a light-emitting source in a subject. In practicing themethod, the light emission intensity from the subject at two or moredifferent wavelengths between about 400 and about 1000 nm is measured.The depth of the light-emitting source is determined using the measuredlight intensities and information related to the optical properties ofthe subject's tissue, e.g., coefficients of attenuation and diffusion inthe subject.

[0024] Information relating to optical properties, e.g., thecoefficients of attenuation and diffusion in the subject, may beobtained by direct measurement of the coefficients in material that isthe same or similar to that of the subject.

[0025] Alternatively, optical-properties information may be obtainedindirectly, by determining, at two or more different wavelengths, lightintensities from a light-emitting source located at each of a pluralityof depths in tissue or material corresponding to that of the subject.The desired depth determination is then carried out by matching themeasured light intensities with the light intensities determined at eachof the plurality of depths.

[0026] In the latter approach, the spectral profile of light intensitiesfrom the light-emitting source may be compared (matched) with each of aplurality of spectral profiles of light intensities from alight-emitting source at each of a plurality of depths.

[0027] These and other objects and features of the invention will becomemore fully apparent when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1A is a cross-sectional view of an apparatus constructed inaccordance with the invention, for use in investigating the location andsize of a light-emitting source in a subject;

[0029]FIG. 1B illustrates features of the apparatus in FIG. 1A inschematic form;

[0030]FIG. 2 is a flowchart of the general steps that can be carried outby the apparatus, in practicing the method of the invention;

[0031]FIGS. 3A is a surface light-intensity image from a light-emittingsource in an animal subject.

[0032]FIG. 3B is a light emission profile from the animal subject inFIG. 3A, showing the curve fit between the measured emission profile(solid lines) and a light-emission profile calculated from a diffusionmodel;

[0033]FIGS. 4A and 4B are plots of the emission spectra of bacterialluciferase in vitro (solid line), and in vivo (dashed lines) at variousdepths in a mouse thigh, as indicated, where the plot in FIG. 4B hasbeen scaled to show the peak intensity in vivo in the region between600-700 nm;

[0034]FIG. 5 shows various locations in an animal at which transmissionspectra were obtained.

[0035]FIGS. 6A and 6B show transmission spectra through a liveexperimental animal at the various animal locations shown in FIG. 5;

[0036]FIG. 6C illustrates a double integrating sphere apparatus usefulfor measuring optical properties of a sample, e.g., tissue;

[0037]FIG. 6D illustrates the experimental set up of a radial fiberprobe, useful for measuring optical properties of tissue in vivo;

[0038]FIGS. 7A and 7B are plots of the absorption coefficients;

[0039]FIGS. 7C and 7D are plots of the isotropic or reduced scatteringcoefficients of various tissues as a function of wavelength;

[0040]FIG. 8A illustrates components of the apparatus used for absolutecalibration determination;

[0041]FIG. 8B illustrates how absolute calibration allows determinationof cell count from an animal subject;

[0042]FIGS. 9A and 9B illustrate how photons diffuse through tissue (9A)and how the photons that diffuse to the surface of the tissue arecaptured in the invention (9B);

[0043]FIG. 10A shows a Monte Carlo simulation following a photon througha “random walk” through turbid tissue;

[0044]FIG. 10B illustrates a calculated light intensity spatial profilecalculated for a light source at 4 mm depth from the diffusion model(solid line) and with Monte Carlo simulations (“+” symbols);

[0045]FIGS. 11A and 11B are plots calculated from the diffusion modelshowing peak intensity (11A) and spot width (FWHM) (10B) as a functionof source depth;

[0046]FIG. 12A is a surface light-intensity image from a light-emissionsource in a subject, and shows the horizontal and vertical profile linesalong which light-intensity profiles were measured;

[0047]FIGS. 12B and 12C are light-intensity profiles measured along thehorizontal and vertical profile lines shown in FIG. 12A;

[0048]FIG. 13A shows spectral image plots of luciferase light-emittingsources acquired in vitro (solid line), from the subcutaneous site shownin FIG. 13B (squares); and from the lung sites seen In FIG. 13C(diamonds).

[0049]FIGS. 13B and 13C are surface light-intensity images from animalswith a subcutaneous light-emitting source (FIG. 13B) and light-emittingsources in the lungs (FIG. 13C).

DETAILED DESCRIPTION OF THE INVENTION

[0050] I. Definitions

[0051] Unless otherwise indicated, the terms below are defined asfollows:

[0052] A “biophotonic function” is a mathematical function thatdescribes a measurable photonic output, such as a light emissionprofile, spectral intensity distribution, or integrated light intensity,in terms of photonic-source variables, such as source depth, size andshape, number of light-emitting cells, wavelength-dependent scatteringand absorption coefficients for specific types of tissue, and spectralcharacteristics of the light-emitter. The photonic function can bebased, for example, on a photon-diffusion model, as discussed below, ona Monte-Carlo simulation, or finite element analysis.

[0053] “Turbid tissue” is non-transparent tissue in a subject that hasboth light scattering and light absorption properties.

[0054] “Target region” refers to an internal subsurface region in asubject, such as a subsurface tissue or organ, a solid tumor, or aregion of infection, that is (i) localized within the subject, (ii)separated from the surface region of the subject by turbid tissue, and(iii) preferably possesses at least one distinguishable feature, such asa tissue or organ-specific antigen, gene or gene product, either as mRNAor expressed protein, a product resulting from activation, deactivation,or regulation, of a cell, organ, or tissue.

[0055] Light-emitting source refers to a source of light emission at thetarget region. The source may itself emit visible light, such as whenthe target tissue is genetically modified to produce recombinantlight-emitting protein when specific genes are expressed by someactivation means. In preferred embodiments, different types oflight-generating proteins, such as bioluminescent proteins (e.g.,luciferases) and fluorescent proteins (e.g., GFP, dsRed, and YFP) areemployed. Exemplary light-emitting sources include prokaryotic andeukaryotic cells transformed with a light-generating protein andadministered to the animal subject, as well as light-producing cellswhich are intrinsically part of an animal made transgenic for alight-generating reporter gene.

[0056] Alternatively, the source may include fluorescent or othermolecules that can be excited by electromagnetic radiation to emitvisible light. For example, a composition effective to cause a target toemit light may be administered to the subject and allowed to localize atthe target location wherein the composition will then emit light, orcause some other moiety in or adjacent the target to emit light. Forexample, the administered composition may be a compound which activatescells in or adjacent the target, and in-turn, causes such cells to emitlight, hence, the localized cells are the target within the subject'sbody region. In yet other embodiments, infectious cells are administeredto a subject, and the progress and locality of the disease aredetermined. The target, in this embodiment, is the cluster of cells at agiven point having a location within the body region of the subject. Inother embodiments of the invention, a body region may have multipletargets, and a subject may have multiple body regions, each regionpotentially having a target.

[0057] “Fluorochromes” or “fluorophores” are molecules which are excitedat or effectively near an excitation maxima, and emit or fluoresce lightat longer wavelengths. The difference between the excitation maxima of afluorochrome, and the emission maxima of the fluorochrome, is known as aStokes shift. The greater the Stokes shift for a given fluorochrome, thegreater the difference in between the excitation spectrum, or range ofwavelengths, which excites the fluorochrome, and the emission spectrumor range of wavelengths of light emitted by the fluorochrome when isfluoresces. Certain embodiments of the invention employ fluorochromeswith high Stokes shifts such with Texas Red, rhodamine, Cy3, Cy5, Cy7,and other deep, far red, or near infra-red (NIRF) fluorochromes.Particularly preferred fluorochromes emit light at wavelengths longerthan about 600 nanometers. Other preferred fluorochromes will emit lightin ranges shorter than 600 nanometers. In particular, variants of “greenfluorescent protein” (GFP), each having various emission spectra, areparticularly useful because, like luciferase, such proteins may besynthesized by the target or cells adjacent or associated with thetarget, or which localized at the target, for example, as in aninfection. GFPs may further be administered as a conjugate to anotherprotein or biological material that localizes at the target or targets.

[0058] In preferred embodiments of the invention, each light-emittingsource, under known light-emitting conditions, has a known emissionspectrum. Each emission spectrum is preferably constant throughout thedetection step. In particular, the relative intensity of light emissionat each wavelength of light remains relative to the intensity of lightemission at all other wavelengths of light within the emission spectrumof the light-emitting source, as the overall intensity of the spectrumchanges. Some embodiments of the invention minimally require at leasttwo distinguishable ranges of wavelengths of emitted light. Morepreferably, particular embodiments of the invention require that eachrange of wavelengths of light behave differently, as a function of thedistance or depth for which such light travels through the body region,from other ranges as they travel through a selected body region of asubject. For example, the intensity of a first range of wavelengths oflight is cut in half for every centimeter of a body region the lighttravels, whereas a second range of wavelengths of light is cut by threefourth for every centimeter of body region the light travels through.The intensity of light having both ranges of wavelengths, where eachrange has the same intensity, will diminish by one half at the firstrange, and three quarters at the second range, after passing through onecentimeter of tissue. By comparing the resulting 2:1 ratio ofintensities from the initial 1:1 ratio of intensities at tissue depthzero, a determination that the light traveled one centimeter through thetissue can be made.

[0059] The invention may be practiced using different light-emittingsources such as different luciferase enzymes. For example, the lightemission from “blue” bacterial luciferase (Photorhabdus luminescens)expressed on agar was analyzed by a spectrometer that revealed aspectrum centered about 485 nm. A “green” firefly luciferase (Photinuspyralis) expressed in PC-3M cells in PBS solution emitted lightdemonstrating a spectrum centered about 570 nm. A “red” luciferaseexpressed in PC-3M cells suspended in PBS solution demonstrated aspectrum centered about 620 nm.

[0060] II Apparatus and Method

[0061]FIG. 1A is a cross-sectional view of an apparatus 20 which may beused in investigating the location and size of a light-emitting sourcein a subject, in accordance with the invention. The apparatus generallyincludes a light-tight enclosure or chamber 22 within whichlight-emission events from the subject can be detected. Light tight box22 may be constructed as described in co-owned PCT publication number WO200163247, which is herein incorporated by reference in its entirety.Briefly, the chamber is defined by back and side walls, such as backwall 21, and has a front opening (not shown, which can opened toprovided access to the interior of the chamber, and closed to provide alight-tight seal. Although light tight box 22 was designed for smallanimals, the technology can also be applied to larger mammals, includinghumans.

[0062] Contained within the chamber is a stage 24 on which the subjectis placed and preferably immobilized during light-emission detection.Although many of the examples described herein have to do with a smallmammalian subject, typically a mouse or a rat, it will be appreciatedthat the same principles and methods will apply to other animals as wellas human subjects, employing suitable scale-up in chamber size and wherenecessary, suitable increases in the size and number of light emittersin the target region. The stage in the apparatus shown is designed to beraised and lowered to a selected vertical position within the chamber bya conventional wormscrew mechanism shown generally at 26 and under usercontrol. The stage position within the chamber may be monitored by anyof a number of known tracking devices that do not require visible lightsignals.

[0063] The viewing optics includes to two general components. The firstis a lens assembly 28 that serves to focus light-emission events fromthe surface of the subject onto the detection screen of a photodetector32, which is typically a photodetector pixel array of a charged-coupleddevice that can be operated in a cooled condition to minimize intrinsicnoise level. The construction of the lens assembly and its opticalcoupling to the detector is conventional. One preferred CCD is a model620 CCD, commercially available from Spectral Instruments.

[0064] The second, and optional, component of the optical system is awavelength filter wheel 30 containing a number of optical bandpassfilters designed to block light transmission in all but a selected rangeof visible-light wavelengths. One standard installation includes 3filters; a shortpass filter 30 a, for wavelengths less than 510 nm (bluelight filter), a midpass filter 30 b having a bandpass in the 500-570 nmrange (green light filter), and a longpass filter for wavelengthsgreater than 590 nm (red light filter). In some applications, filterscan provide more precise bandpass, e.g., every 20 nm. The just-describedviewing optics and detector are also referred to herein, collectively,as an optical system.

[0065] The apparatus also includes a control unit 34. Varioususer-controlled settings, such as stage height, orientation, andtranslation position, bandpass filter, and detector mode are madethrough a control input pane; 36 in the apparatus.

[0066]FIG. 1B shows a more schematic representation of apparatus 20,here shown in a scale for use in imaging a small mammalian subject, suchas a mouse 38. The lens assembly is represented here as a single lens28, the filter wheel at 30, and a CCD detector at 32. The detectoroutput is supplied to a computational unit 40 in the control unitcomputational unit 34 which includes a processor 40 and a storage unit42 for data files. The content of the data files, and operation of thecomputational unit, in response to light-detection signals from thedesigns from detector 32 will be described below. The control unit isoperatively connected to a display/output device 44, such as a computermonitor, for displaying information and images to the user.

[0067] The operation of the computational unit can be appreciated fromthe method of the invention carried out by the apparatus, which will besummarized with respect to FIG. 2, and detailed further below. The firststep in the method is to obtain a first measured light-intensityprofile. This is done by first localizing a light-emitting source withina subject. This may be done, as noted above, in a variety of ways,including introducing into the subject, a gene effective to produce alight-emitting protein, such as luciferase, in the target region, orlocalizing a fluorescent compound at the target source, both accordingto methods which are known, e.g., as described in co-owned PCTapplications WO99US/30078, WO00US/7296, WO99US30080; and WO97US6578, allof which are incorporated herein by reference.

[0068] The subject so prepared is placed in the light-tight chamber, andlight-emission form the subject surface originating from light-emissionevents at the target region are measured, digitized, and placed in thecomputational unit, as at 52 in FIG. 2. The computational unit uses thisdata to generate a spatial profile of light emission, as at 54. Thisprofile could be taken along a selected row and a selected column ofdetector elements in the detector representing profiles along x(horizontal) and y (vertical) axes in a plane parallel to the stage inFIG. 1A. That is, the profile is a plot of measured intensity valuesalong a selected row and column of detector elements, and represents thedistribution of light intensity values that are focused from the subjectsurface onto the detector array. Exemplary light-intensity valuesmeasured from a subject are shown in solid lines in FIG. 3B. FIG. 3Ashows the emission contours, and FIG. 3B shows the vertical profile, asdetermined at detector pixels corresponding to those positions.

[0069] Turning back now to FIG. 2, the spatial profile or profiles soobtained are matched or fitted with profiles stored in a database 58 ofparameter-based photonic functions which are either empirical functionsmeasured from light-emitting sources of known size and depth, or arecalculated from photon-diffusion models using depth and size parameters,and optionally, other parameters, as will be described below.

[0070] From the optimal curve match, the program can make an initialestimated determination of depth and/or size and/or brightness,preferably all three, of the light-emitting source. According to afeature of the invention, the method is now carried out in a way thatthe depth and size determinations are refined by additional data thatmay be in the form of additional light-intensity data, and/or additionalmodeling data. For example, and as indicated in FIG. 2, the data may bein the form of:

[0071] (a) 2nd-view data, as at 62, meaning light-intensity data,typically spatial profile data, obtained by viewing the subject from asecond view angle. Typically, the subject in the apparatus is tiltedwith respect to the optical system so that light-emission is obtainedfrom a second region of the subject surface;

[0072] (b) Spectral data, as at 64, meaning light-intensity dataobtained at one or more bandpass ranges, e.g., in a blue-light,green-light, or red-light spectral range;

[0073] (c) Transmittance data, as at 66, meaning predetermined-lightintensity values obtained typically as a function of wavelength atselected locations in a subject that approximates the subject ofinterest;

[0074] (d) Tissue properties data, as at 68, meaning predeterminedvalues of tissue parameters, such as reduced scattering coefficientμ′_(s), absorption coefficient μ_(a), or effective coefficient μ_(eff)corresponding to the subject tissue through which light is diffusing.This data is used, for example, to refine the spatial profile curvesgenerated by a diffusion model;

[0075] (e) Simulation data, as at 70, meaning predeterminedlight-intensity values, typically spatial profile data, obtained byplacing a light-source of known intensity and size at selected locationsin a model representing the subject target region and adjacent surface,or in an actual subject that approximates the subject; and

[0076] (f) Total intensity data, as at 72, meaning total light intensitysummed or integrated over an entire spatial profile or an entiredetector array. The program run by the computation device receives theadditional information at 60, and uses the information to refine thedepth and size determination of the light-emitting source, at 74, aswill be considered below. Finally, the refined determination of sourcedepth, size, and optionally, shape and number of cells in thelight-emitting source, is displayed to the user, either in the form ofdata and/or one or more subject images showing the location andintensity of the light-emitting source.

[0077] (g) Absolute calibrated light intensity, as at 73. Calibration toabsolute intensity allows one to convert counts/sec/pixel in the CCD toradiance photons/s/cm²/sr (sr=steradians). FIG. 8A illustrates anoptical assembly 100 used in the calibration method. The assemblyincludes, in addition to a CCD detector 102, lens 104 (representing alens assembly, as above), a bandpass filter 106 and a low-light-levelintegrating sphere, such as a OL Series 425 sphere available fromOptronic Laboratories, which acts as a source of a known lightintensity. The known radiance from the sphere, measured inphotons/sec/cm²/sr, allows one to calculate the calibration factor thatrelates the two numbers.

[0078]FIG. 8B illustrates how absolute measured intensity is used tocalculate total number of cells in a light-emitting source. It isassumed that each cell will produce a photon flux φ_(c)photons/sec/cell. The radiance from a light-emitting source 110 in asubject 112, as measured by detector 102, measures the number ofphotons/s/cm²/sr. The measured radiance is then integrated over a regionof interest to convert to a source flux φ_(s) photons/sec. From theknown flux values, the number of cells in the light-emitting source isreadily calculated as φ_(s)/φ_(c).

[0079] III. Light-Emission Data and Parameters

[0080] This section considers various types of light-emission data thatmay be collected in practicing the invention, and discusses how the datamay be used in photon-diffusion modeling, and/or for determining depthor size of a light-emitting source based on curve matching with a modeldiffusion curve.

[0081] Light-intensity spatial profile. The basic light-emission datathat is determined is a light-intensity spatial profile which has beendescribed above, with reference to FIG. 3B. A measured light-intensityspatial profile shows the spatial distribution of light intensityemitted from the surface region of subject. The light-intensity spatialprofile may be obtained along a single line, along 2 or more lines(e.g., along the x and y directions with respect to the stage supportingthe subject). The entire 2D surface distribution of light may also beused. A modeled spatial profile represents the predicted light-intensityspatial distribution calculated from a model of photon diffusion througha turbid tissue. By matching a measured profile with a modeled profile,an approximate depth and/or size of light-emitting source can bedetermined.

[0082] Total light intensity. Total light intensity is the total lightintensity summed or integrated over all of the detector elements in thedetector. Total light intensity can be used as a further constraint onthe fitting model used to determine source, location, and brightness.

[0083] Absolute calibrated light intensity. As described above withreference to FIG. 8B, the absolute light-intensity flux can be used tomeasure total flux from the light-emitting source. Assuming that thissource is made up of cells which each emit an average cell flux φ_(c),the total number of light-emitting cells making up the source can bedetermined. This determination, in turn, can be used to refine the totalsource mass or volume determination, based on a known or estimated cellmass ratio for the tissue source.

[0084] Spectral data. Spectral data is light-intensity data collected atparticular wavelength ranges, e.g., the blue-light, green-light, andred-light wavelength ranges noted above. Because light is absorbedpreferentially at shorter wavelengths by subject tissue, andparticularly by hemoglobin in subject tissue, the relative intensitiesof light at different wavelengths, can provide information about sourcedepth and can be used in producing refined wavelength-dependent spatialprofiles, based on wavelength-dependent tissue scattering and absorptioncoefficients.

[0085]FIGS. 4A and 4B depict the differential effect distance or depthhas upon light that travels through the tissue of the body region, atdifferent wavelengths or ranges of wavelengths. The graph showsincreasing light intensity on the y-axis and increasing light wavelengthon the x-axis. The solid line in FIG. 4A represents the in vitroemission spectra for the light-emitting source that correlates to azero-depth emission spectrum which comprises a broad single peakcentered about 495 nm. The broad dashed line represents the emissionspectrum of the light-emitting source after it has passed through aboutone millimeter of tissue (in vivo). At about 600 nm, the one millimeterdepth emission spectrum develops a small peak, as the once large peak atabout 495 nm begins to diminish in relative intensity. The intermediatedashed line represents the emission spectrum of the light-emittingsource after it has passed through about two millimeters of tissue (invivo). The 495 nm peak becomes lesser in intensity, while the 600 nmpeak increases and becomes more pronounced. Lastly, the dotted linerepresents the emission spectrum of the light-emitting source after ithas passed through about four millimeters of tissue (in vivo) where the495 nm peak has been further reduced.

[0086]FIG. 4B represents the emission spectrum at four millimeters depthof tissue scaled against the zero tissue depth emission spectra. The 600nm peak is prominent over the once prominent 495 nm peak. In thisexample, it is the ratio of the 600 nm peak to the 495 nm peak thatprovides information about the depth of the light-emitting source.

[0087] Preferred methods of the invention employ self-scalingcomputations so that the depth of the light-emitting source may bedetermined independent of its overall intensity. To achieve this end,functions are formulated that utilize ratios of two or more ranges ofwavelengths, where each range of wavelengths travels through tissue in abody region at different rates as a function of depth of the target fromthe surface of the body region.

[0088] Through-body transmittance. Through-body transmittance ismeasured by placing a know-intensity light-source at a selected locationagainst or inside a subject, and measuring the light-intensity that istransmitted through the subject on multiple surfaces. The transmittancewill have a strong spectral component, owing to the strong spectraldependence of the scattering and absorption coefficients of differenttissues (see below). Typically, transmittance values are pre-determinedusing a model subject, by measuring total transmittance at variousselected locations and at each of a number of spectral ranges.

[0089]FIG. 5 shows a top view of a subject having a plurality of bodyregions indicated by numbers 1-16. In this example, the subject is animmobilized mouse, and each body region corresponds to pre-determinedregions of interest. Prior to depth determinations by the practice ofthe methods of the invention, the subject is optically analyzed todevelop a data file of optical characteristics for each body region. Inthis example, each body region is placed between a spectral light sourceand a spectral detector. Light transmission spectra are obtained foreach body region. The result is analogous to placing each body region ina spectrophotometer cuvette and measuring the spectral profile of eachregion at different wavelengths and wavelength ranges, to create a datafile of how light transmits through different body regions, and thetissues within such regions. The device of FIG. 1 may be used to developsimilar data for particular ranges of wavelengths provided the device isadapted to trans-illuminate, with respect to the detector, the subjectat each body region tested. Thus, the device of FIG. 1 becomes, inessence, a whole animal spectrophotometer.

[0090]FIGS. 6A and 6B show the empirical spectral data gathered from thespectral analysis described immediately above. As is described below inmore detail, this data, either as whole spectra, or particular rangeswithin each spectrum, can be used to create the data files forcomparison with detected intensity information, or for developingfunctions to compute target depth.

[0091] In a more detailed approach, the apparatus provides a whole bodyscanner for scanning an entire subject, or portions thereof, whichfurther comprises a scanning two-color laser for scanning the body of asubject. The scanner moves through an arc laser light of at least twocolors, either simultaneously, or sequentially. As the laser light beamsupward, it penetrates the body of a subject placed within a rotationaltube. The detector placed opposite the laser light source is adapted toreceive and measure the laser light as it moves through its arc path.

[0092] Tissue optical properties. There are two important opticalproperties of subject tissue used in photon diffusion modeling. Thefirst is the absorption coefficient μ_(a) of the tissue, which isrelated to the fraction of incident light that is absorbed per unitpathlength. As seen in FIGS. 7A and 7B, which plot absorptioncoefficients for a variety of tissue types over the visible spectrum,the absorption coefficient is highly dependent on the nature of thetissue, and the wavelength of light, with each tissue showing a peakabsorption in the 500-600 nm range. The relatively low absorptioncoefficient above about 600 nm is consistent with the spectral data seenin FIG. 13A, and with the total transmittance data shown in FIGS. 6A and6B.

[0093] The second important optical property is the reduced scatteringcoefficient μ′_(s) of the tissue, which is the fractional decrease inintensity per unit length of penetration of light due to large anglelight scattering. As seen in FIGS. 7C and 7D, which plot reducedscattering coefficient for the same tissues over the visible spectrum,scattering effects can be relatively different in different tissues. Ingeneral, the reduced scattering coefficient decreases somewhat at longerwavelengths.

[0094] A double integrating sphere (DIS) system is a widely used tool tomeasure the optical properties of tissue or any turbid medium since itconveniently measures the diffuse reflectance, R_(d), and the diffusetransmittance, T_(d), simultaneously. FIG. 6C illustrates the doubleintegrating sphere apparatus 74 used to measure these values in order toobtain optical properties such as the absorption coefficient, μ_(a), andthe reduced scattering coefficient, μ′_(s) (Prahl, S. A., et al.,Applied Optics 32:559-568 (1993); Pickering, J. W., et al., AppliedOptics 32:399-410 (1993)). The sample 76 is placed between the twointegrating spheres 78 and 80, with internal baffles 82 positioned toprevent measurement of directly reflected or transmitted light. Thespheres are coated with a highly reflective material to minimizeabsorption of light. Light is detected with detectors 84 and preferablyanalyzed using a computer 86. Sample illumination between 400-1000 nm isachieved using an arc lamp connected to a monochromator 88, via a fiberoptic cable 90 and lens 92.

[0095] From diffuse reflectance, R_(d), and diffuse transmittance,T_(d), values, the absorption and the reduced scattering coefficientsare obtained using the inverse adding-doubling program(http://omic.ogi.edu/software/iad/index.html). This program is anumerical solution to the one speed radiative transport equation, whichdescribes light propagation at steady state in a scattering medium(Prahl, S. A., et al., Applied Optics 32:559-568 (1993)). The program isan iterative process, which estimates the reflectance and transmittancefrom a set of optical parameters until the calculated reflectance andtransmittance match the measured values.

[0096] A characteristic of the DIS system is the fact that tissues needto be extracted before the measurement. Therefore, it is desirable tomaintain tissue viability under measurement conditions. Vasculardrainage of hemoglobin and tissue hydration need to be considered,especially at wavelengths where hemoglobin and water absorption arehigh. Partly in view of the foregoing, a preferred method of measuringtissue optical properties is a non-invasive in vivo measurement, asfollows.

[0097] A radial fiber probe similar to that described by Bevilacqua(Bevialcqua, F., et al., Applied Optics 38:4939-4950 (1999)) may be usedto measure optical properties non-invasively or with minimalinvasiveness in vivo. FIG. 6D illustrates an experimental set up of aradial fiber probe 94. Fiber probe 94 includes an illumination source95, at least one illumination fiber 96 and several (e.g., six) detectionfibers 97 generally within a 1-2 mm distance. The probe is then placeperpendicularly on a tissue of interest 99, preferably with minimalcontact pressure. The output of the detection fibers 97 is fed into aset of corresponding detectors 98. The optical properties are spatiallyresolved from intensity versus radial distance from the source. At smallsource-to-detector separations, simple analytical models such as thediffusion approximations are not well suited to describe the systemtherefore Monte Carlo methods are typically used to analyze the data.

[0098] IV. Photon Transport Models

[0099] This section considers photon-transport models for simulatingphoton emission at the surface of a body from a light source below thesurface and moving through a turbid medium. In particular, it is desiredto generate simulated light-intensity spatial profiles based on (i)depth of the light-emitting source, (ii) size of the light-emittingsource, and (iii) light-absorption and light-scattering coefficients.Since the light-absorption and light-scattering coefficients aredependent both on wavelength and on the nature of the tissue throughwhich the photons diffuse, the model may be refined to specificallyconsider the nature of the tissue and the spatial profiles at selectedwavelengths.

[0100] The photon-diffusion model employed herein makes a number ofsimplifying assumptions for purposes of generating an initiallight-intensity spatial profile from which initial source-depth andsource-size information can be generated, as will be considered inSection V. The model may then be expanded by considering additionaltissue-dependent and/or wavelength dependent information to refine thespatial profile, for purposes of improving the curve fit with a measuredspatial profile. Alternatively, the initial depth and size informationcan be refined by other types of curve or data matching. Both approachesfor refining the initial depth/size approximation will be considered inSection V below.

[0101]FIG. 9A shows a photon path as it diffuses through a turbid tissueformed of cells, such as cell 111 containing organelles 113, As seen,the cell size, typically in the 20-30 micron size range, is largerelative to the wavelength λ of light, which is between about 0.4 and0.6 microns. Scattering is due to abrupt discontinuities (<<λ) atmembranes and is characterized by an inverse length scatteringcoefficient μ_(s) of about 10-20 mm⁻¹. The scattering anisotropy g isabout 0.9, which gives a reduced scattering coefficientμ′_(s)=μ_(s)(1−g), or about 2 mm⁻¹. The absorption coefficient μ_(a) (λ)is between 0.01 and 1 mm⁻¹, and as seen above, has a strong wavelengthdependence. The absorption and scattering coefficients arewavelength-dependent, but for λ>˜600 nm, it is generally true thatμ_(a)<<μ′_(s) in tissue.

[0102] A quantitative description of transport of light from a targetsite within a body to a light detector located adjacent a body surfacemay be achieved by a solution of a radiative transfer diffusion equationof light with appropriate boundary conditions (e.g., R. C. Haskel; et alin “Boundary conditions for the diffusion equation in radiativetransfer”, J. Optical Soc Am., 11A:2727 (1994)). One approach, forexample, uses an extrapolated boundary condition shown schematically inFIG. 9B in which optical fluence vanishes at a planar surface that isdisplaced from the physical boundary at a distance z_(b) that takesaccount of the Fresnel reflection of light at the physical boundary.

[0103] As a first approximation, the body in which the light-emittingsource is located is represented as a semi-infinite, homogenous turbidmedium that is both scattering and absorbing. The approximation isillustrated in FIG. 9B which shows a light-emitting point source 114 inslab 116 a distance d below the slab surface. The diffusion equation is

D∇ ²φ(r)=μ_(a)φ(r)−S(r)  (1)

[0104] and Fick's law is

j(r)−D∇φ(r)  (2)

[0105] where φ is the isotropic fluence (watts/m²), j is the smalldirection flux (watts/m²), S is the power density (watts/m³), r is theradius, and $\begin{matrix}{D = \frac{1}{3\left\lbrack {{\left( {1 - g} \right)\mu_{s}} + \mu_{a}} \right\rbrack}} & (3)\end{matrix}$

[0106] The Green's function solution for a point source P (watts) is$\begin{matrix}{{\varphi (r)} = {\frac{P}{4\pi \quad D\quad r}{\exp \left( {{- \mu_{eff}}r} \right)}}} & (4)\end{matrix}$

[0107] where μ_(eff)={square root}{square root over(3μ_(a)(μ′_(s)+μ_(a)))} and μ′_(s)=(1−g)μ_(s).

[0108] The solution to the diffusion equation in slab geometry using theextrapolated boundary condition is obtained by summing contributionsfrom the source 114 plus image source 118 (as shown in FIG. 9B),resulting in the following equation for radiance at the surface (viewingperpendicular to the surface) for a point source P (watts) at a depth d.$\begin{matrix}{L_{z = 0} = {\frac{1}{4\pi}\left( \frac{P}{4\pi \quad D} \right)\left\{ {\frac{\exp \left( {{- \mu_{eff}}r_{1}} \right)}{r_{1}} - \frac{\exp \left( {{- \mu_{eff}}r_{2}} \right)}{r_{2}} + {3{D\begin{bmatrix}{{\frac{d}{r_{1}^{2}}\left( {\mu_{eff} + \frac{1}{r_{1}}} \right){\exp \left( {{- \mu_{eff}}r_{1}} \right)}} + {\frac{d + {2z_{b}}}{r_{2}^{2}} \times}} \\{\left( {\mu_{eff} + \frac{1}{r_{2}}} \right){\exp \left( {{- \mu_{eff}}r_{2}} \right)}}\end{bmatrix}}}} \right\}}} & (5)\end{matrix}$

[0109] where r₁={square root}{square root over (ρ²+d²)}, r²={squareroot}{square root over (ρ²+(d+2z_(b))²)} ρ is the radius on the slabsurface and $\begin{matrix}{z_{b} = {\frac{1 + R_{eff}}{1 - R_{eff}}\frac{2}{3\left( {\mu_{a} + \mu_{s}^{\prime}} \right)}}} & (6)\end{matrix}$

[0110] and R_(eff) is the effective reflection coefficient which isabout 0.43 for tissue.

[0111] Plotting surface radiance as a function of ρ gives the plot shownin the solid line in FIG. 10B, which has the same derivation as thelight-intensity profile plot shown in dotted lines in FIG. 3B.

[0112] The spatial profile curve for a light-emitting point in a turbidmedium was also calculated using a much more computationally intensiveMonte Carlo simulation, which follows each photon through a random walk,as illustrated in FIG. 10A. The Monte Carlo simulation, shown in “+”symbols in FIG. 10B closely matches the spatial profile calculated fromthe diffusion equation.

[0113] To illustrate how the diffusion equation may be used to modelphoton diffusion through various tissues and a various depths, thespatial profile was calculated for various values of μ_(eff),corresponding to μ_(a) and μ′_(s) values of 2.0 and 20 cm⁻¹,respectively, for μ_(eff)=11 cm⁻¹; 0.4 and 10 cm⁻¹, respectively, forμ_(eff)=3.5 cm⁻¹, and 0.05 and 5 cm⁻¹, respectively, for μ_(eff)=0.87cm⁻¹. The three values of for μ_(a) correspond roughly to tissueabsorption for blue, green, and red wavelengths.

[0114] The plots in FIGS. 11A and 11B show that intensity decreases andspot width increases with increasing depth, and that large values ofμ_(eff) result in large attenuation of peak intensity with narrower spotwidth.

[0115] V. Determining Depth and Size Information

[0116] In practicing the method of the invention, the subject isinitially treated to localize light-emitting molecules at a targetsource, as detailed above. The subject is then placed at a selectedlocation within the light-tight chamber of apparatus 20 (appropriatelyscaled for subject size) and the optical system is adjusted to measurelight emission events from the source at the surface region of thesubject between the light-emitting source and the detection optics. Withthe subject immobilized at a selected optical perspective, alight-intensity spatial profile of surface light emission is obtained,also as described above. FIG. 3A is a surface map of measured lightintensity from a subsurface light source, which was subsequently used togenerate the spatial profile shown in FIG. 3B.

[0117] The spatial profile(s) are then fitted with parameter-basedbiophotonic functions that relate light-emission intensity to depth of alight-emitting source (and in some cases, source size) to obtain aninitial determination of source depth (and in some case, source size).One preferred biophotonic function is that derived from the simplifieddiffusion model discussed in Section IV above, in which light intensityfrom a point source or source having a defined spherical or ellipsoidalvolume is calculated as a function of source depth, fixed scattering andabsorption coefficients, and surface distance r from the source.Conventional non-linear least-squares curve fitting techniques, such asLevenberg-Marquardt (“Numerical Recipes in C”, Press et al., eds,Cambridge Press, NY, 1989). are suitable for the curve fitting. Curvefitting can be done using a single 1-dimensional (“1-D”) profile, asshown in FIG. 3, a plurality of such profiles, or the entire 2-D spatialdistribution (e.g., as shown in FIG. 12A). Curve-fitting calculations tothe data shown in FIG. 3B (dashed line) indicate a depth of 2.7 mm,which compares well with the estimated actual depth of 2.2 mm.

[0118] FIGS. 12A-12C give another example of how depth and sizeinformation can be determined from spatial profiles. The light-emittingsource here is a subcutaneous elliptical tumor having actual dimensionsin the horizontal and vertical directions of 2.7 mm and 3.2 mm,respectively, and a tumor thickness of 1.5 mm. The horizontal andvertical profiles were (FIGS. 12B and 12C, respectively) were fittedwith curves generated for an ellipsoid light-emitting source havinghorizontal and vertical dimensions of 1.3 and 2.4 respectively, athickness of 1.5 mm, and a depth of 0.4 mm.

[0119] The source depth (and optionally, source size) determination fromthe initial curve fitting can be refined, and source size approximated,by employing additional data whose effect is to refine the parameters ofthe biophotonic function. The nature of the additional data, and themanner in which it refines the parameters, e.g., depth and size, of thebiophotonic function, providing refined depth and source-sizeinformation, will now be considered for each of the information typesoutlined in Section II above, with respect to FIG. 2.

[0120] A. 2nd-view information. To obtain 2nd-view data, the subject isrotated with respect to the viewing and detection optics for viewing thelight-emitting source from another perspective. From this secondperspective, a second light intensity profile is obtained, providing asecond depth determination with respect to a second subject surfaceregion. By determining the intersection of the source depths at twodifferent surface locations, a more accurate depth and/or source sizecan be determined. The more views that are obtained, the more accuratelydepth and source size can be determined.

[0121] B. Spectral data. A target's depth within a body region may bedetermined, or the target depth refined, by (i) causing the target toemit light at two or more wavelength ranges, and (ii) comparing thedifferences in each range's light transmission through the body region,at a surface of the body region, provided that the light in eachwavelength range transmits through the body region differently from thelight in other wavelength ranges as a function of depth.

[0122] Typical spectral curves (representing total light intensity atthe indicated wavelengths) as a function of source depth are shown inFIG. 4A, discussed above. As seen particularly in FIG. 4B (where thepeaks are scaled to the in vitro spectral curve), the ratio of peakheight at above 600 nm, e.g., 620 nm, to that the in vitro (zero-depth)peak height, e.g., around 500 nm, increases dramatically as a functionof depth. Thus, by generating a spectral curve and determining the ratioof peak heights, typically above and below 600 nm, e.g., 620 nm: 500 nm,accurate depth information based on the wavelength-dependent loss inintensity in turbid tissue, can be determined.

[0123] A useful formula for determining approximate depth from spectralmeasurements can be derived from Eq. 4 as follows: $\begin{matrix}{d \approx \frac{\ln \left( {\varphi_{1}{D_{1}/\varphi_{2}}D_{2}} \right)}{\mu_{eff2} - \mu_{eff1}}} & (7)\end{matrix}$

[0124] where d represents depth, φ represents the measured lightintensity, μ_(eff) represents the empirically determined effectivecoefficient of attenuation, and D is the empirically determineddiffusion coefficient. The subscripts 1 and 2 in the above formula referto two separate wavelengths at which measurements are made.

[0125] Using the above formula, depth may be determined from two or morelight intensity measurements at different wavelengths or ranges ofwavelengths. In one embodiment, the depth may be obtained by executingthe following series of steps: (1) Image the bioluminescent cells invitro as well as in vivo (in the animal) at two wavelengths; (2)Quantify the images by measuring, e.g., the peak intensity or theaverage (integrated) intensity for each image; (3) Calculate the ratioof the in vivo image data to the in vitro image data at each wavelength;and (4) Calculate the depth using Equation 7 and an effective scatteringcoefficient μ_(eff) obtained, e.g., from tissue property measurements.An application of this approach is illustrated in Example 1.

[0126] In one embodiment, it is preferable to have a significantlydifferent value for each μ_(eff). Animal tissue provides such adifference in μ_(eff), due largely to the presence of hemoglobin, whichhas a large absorption peak just below 600 nm, and relatively lowabsorption above 600 nm.

[0127] Alternatively, the additional spectral data may include one ormore spatial profiles taken at one or more selected wavelengths orwavelength ranges. The profile(s) are then compared with model intensityfunctions generated, for example, from a photon diffusion model above,using wavelength-specific values for the absorption, scattering, and/oreffective coefficients, as described above with respect to FIG. 3B.

[0128] In still another application, intensity values can be measured atdiscrete wavelengths, spaced every 20 nm. The spectral plots in FIG. 13Ashow green luciferase in cells in PBS solution, and spectral curves forgreen luciferase localized subcutaneously at a site indicated in FIG.13B and in the lungs as shown in FIG. 13C. All the curves are normalizedto a value of 1 at 700 nm. The ratio of intensities at 560 nm to 620 nmis indicative of depth.

[0129] As noted above, a related aspect of the invention provides amethod of determining the depth of a light-emitting source in a subject.In practicing the method, the light emission intensity from the subjectat two or more different wavelengths between about 400 and about 1000nm. The depth of the light-emitting source is determined using themeasured light-emission intensities, and information related to theoptical properties of the subject, e.g., coefficients of attenuation anddiffusion in the subject.

[0130] Information relating to the optical properties, e.g.,coefficients of attenuation and diffusion in the subject may be obtainedby direct measurement of the coefficients in material that is the sameor similar to that of the subject. This information, combined withmeasured intensities at two wavelengths in vitro (zero depth) and invivo (the depth to be determined) can be applied to Equation 7 todetermine the depth of the light source, as described above, and asillustrated in Example 1.

[0131] Alternatively, optical-properties information may be obtainedindirectly, by determining, at two or more different wavelengths, lightintensities from a light-emitting source located at each of a two ormore of a plurality of depths in tissue or material corresponding tothat of the subject. The desired depth determination is then made bymatching the measured light intensities, e.g., the ratio of theintensities at the different wavelengths, with the light intensitiesdetermined at each of the plurality of depths. In a more specificapproach, the spectral profile of light intensities from thelight-emitting source may be compared (matched) with each of a pluralityof spectral profiles of light intensities from a light-emitting sourceat each of a plurality of depths.

[0132] C. Through-body transmittance data. Through-body transmittancedata is obtained as described above, and illustrated in FIGS. 5, 6A and6B for a small-animal subject. As noted above, the information providesa wavelength dependent light-transmission value through a selectedtissue or tissues of a known thickness. Predetermined transmittance datafrom a number of selected locations in a subject can be used to estimateaverage whole-body scattering and absorption coefficients at thedifferent subject locations, for purposes of refining the modeledspatial profile, either over the entire visible spectrum, or at selectedwavelengths, used for curve fitting to the measured spatial profile.

[0133] D. Tissue properties data. The tissues property data, such asthat shown in FIGS. 7A-7D, typically includes wavelength dependentabsorption and scattering coefficients for each of the major bodytissues. This data is used, for example, in the above model of photondiffusion, to refine the spatial profiles that would be produced bylight transmission through a given tissue, at a given wavelength,typically in the red wavelength. Thus, for example, if thelight-emitting source in the subject is muscle, and the spatial profileis taken in the red wavelength, a refined spatial profile generated withthe tissue-specific and wavelength-specific absorption coefficient willprovide a refined spatial profile curve for curve fitting with themeasured curve.

[0134] E. Simulation data. In another embodiment, a test point orotherwise known-shape light-emitting source may be introduced into ablock or slab that simulates turbid tissue. The block can be preparedwith various scattering and diffusion coefficients, and various shapesto simulate light-emitting source conditions in a subject. Uponplacement of the test point, spectral profiles may be taken from theouter surface of the body region where the test point was placed. Thisdata then is correlated with the actual depth and location of the testpoint. By moving the test point from point to point within the block, aseries of spectral measurements can be made. From this series ofspectral measurements, a data file can be assembled to model thespectral responses of different regions within the subject.

[0135] F. Integrated Light Intensity

[0136] Summed or integrated light-intensity, as noted above, refers tothe light-intensity summed over all or some defined area of the detectorarray. The integrated light intensity can be compared with the integralof Equation 5 to provide yet another estimate of source depth andbrightness. This information can be used in conjunction with the profileinformation. The integrated light intensity can also be calculated formultiple wavelengths.

[0137] G. Calibrated-intensity data. In the absence of calibratedintensity data, intensity measurements may vary from camera to camera,and measured values will depend on variable such as field of view,binning, time, and f-stop.

[0138] Absolute calibrated intensity (Section III above), allows one torefine the depth-of-source determination, based on curve fitting to truepeak value, and to estimate the number of bioluminescent cells intissue, as discussed in Section III above.

[0139] In still other embodiments, the invention provides for theintegration of other imaging data with the intensity and spatialdistribution data to produce greater detail three-dimensional maps ofthe subject and target located therein. For example, compileddifferential light transmission data, such as disclosed above, may beinterlaced with coordinate systems derived from other three dimensionalimaging systems such as stacked slices of MRI images forming a threedimensional digitized “image” or coordinate system. Such a coordinatesystem may then be used to better illustrate the anatomy of the subjectfor which a target is being identified. In the case where the subject isa laboratory animal such as a rat, such animals are highly uniform instructure from one animal to the next for a given strain. Consequently,a coordinate data, spectral data, and spatial intensity pattern filesmay be developed by a commercial vendor, and sold for use withsimplified detection units such as shown in FIG. 1. If modelthree-dimensional information is supplied as a data file, then userunits do not need to be equipped for three-dimensional scanning andmapping. The two-dimensional images contemplated in the methods andapparatuses disclosed above may be combined with the three-dimensionaldata files provided by the vendor to yield complete three dimensionalinformation about the size, shape, depth, topology, and location of atarget within the subject.

[0140] The following examples illustrate, but in no way are intended tolimit the present invention.

EXAMPLE 1 Calculating Depth of a Light-Emitting Object UsingTwo-Wavelength Spectral Images

[0141] The spectral information shown in FIG. 13A was used to calculatethe depth of luciferase-labeled cells in an animal that received asub-cutaneous injection of the cells (FIG. 13B) and an animal havinglabeled cells in its lungs (FIG. 13C). The analysis was performed usingdata at wavelengths equal to 600 nm and 640 nm using the followingsteps: (i) the bioluminescent cells were imaged in vitro as well as invivo at 600 nm and 640 nm; (ii) the images were quantified by measuringthe average intensity for each image; (iii) the ratio of the in vivoimage data to the in vitro image data were determined at eachwavelength; and (iv) the depth was calculated using Equation 7 and anaverage effective scattering coefficient μ_(eff) estimated from theabsorption and scattering coefficients in FIG. 7.

[0142] For the subcutaneous cells, the ratios of in vivo to in vitrointensities (relative intensities, or φ) are 0.35 and 0.75 at 600 nm and640 nm, respectively. For the lung signals, these same ratios are 0.05and 0.47. Using μ_(a)=0.25 mm⁻¹ and μ_(s)′=1.0 mm⁻¹ at 600 nm andμ_(a)=0.05 mm⁻¹ and μ_(s)′=1.0 mm⁻¹ at 640 nm results in μ_(eff)=0.97mm⁻¹ at 600 nm and 0.4 mm⁻¹ at 640 nm. The values of the diffusecoefficient D are 0.27 mm and 0.32 mm at 600 nm and 640 nm respectively.Substituting these numbers into Equation 6, reproduced below,$d \approx \frac{\ln \left( {\varphi_{1}{D_{1}/\varphi_{2}}D_{2}} \right)}{\mu_{eff2} - \mu_{eff1}}$

[0143] (in this case, subscript 1 refers to 600 nm and subscript 2refers to 640 nm), results in d=1.6 mm and d=4.0 mm for the subcutaneousand lung depths, respectively.

[0144] Although the invention has been described with respect toparticular embodiments and applications, it will be appreciated thatvarious changes and modification can be made without departing from theinvention.

It is claimed:
 1. A method of investigating the location and size of alight-emitting source in a subject, comprising: (a) obtaining a firstmeasured light intensity profile constructed by measuring, from a firstperspective with a photodetector device, photons which (i) originatefrom the light-emitting source, (ii) travel through turbid biologicaltissue of the subject, and (iii) are emitted from a first surface regionof interest of the subject; (b) fitting said first measured lightintensity profile with a parameter-based biophotonic function; and (c)refining the parameters of the biophotonic function, using data otherthan said first measured light intensity profile, to obtain anapproximate depth and size of the source in the subject.
 2. The methodof claim 1, wherein said refining includes using data relating to thewavelength of photons emitted from the surface of the subject.
 3. Themethod of claim 2, for use in investigating the depth of thelight-emitting source in the subject, wherein said refining includes (a)measuring light emission intensity from the subject at two or moredifferent wavelengths between about 400 and about 1000 nm, (b) obtaininginformation related to optical properties in the subject's tissue, and(c) using the measured light intensities and the obtained information torefine the depth of the light-emitting source.
 4. The method of claim 3,wherein the steps (a), (b), and (c) above are carried out by a protocolselected from the group consisting of: A. step (b) includes measuringthe coefficients of attenuation and diffusion in tissue or materialcorresponding to that of the subject. and step (c) includes using saidcoefficients in a formula to estimate depth of said source; B. step (b)is carried out by determining, at two or more different wavelengths,light intensities from by a light-emitting source located at each of aplurality of depths in tissue or material corresponding to that of thesubject, and step (c) is carried out by matching the measured lightintensities with the light intensities determined at each of theplurality of depths. C. step (a) includes measuring a spectral profileof light intensities from the light-emitting source, step (b) is carriedout by determining spectral profiles of light intensities from alight-emitting source at each of a plurality of depths, and step (c) iscarried out by matching the measured profile with the determinedprofiles, to identify a best curve fit; and D. step (a) includesmeasuring the light-intensity profile at a different wavelength.
 5. Themethod of claim 1, wherein said refining includes using data measuredfrom said subject.
 6. The method of claim 5, wherein said refiningincludes using data obtained from a second measured light intensityprofile, said second profile being constructed by measuring, from asecond perspective with said photodetector device, photons which (i)originate from the light-emitting source, (ii) travel through turbidbiological tissue of the subject, and (iii) are emitted from a secondsurface region of interest of the subject
 7. The method of claim 5,wherein obtaining said data includes the steps of (i) measuring totallight intensity from the first surface region of interest, (ii)estimating tissue-region depth, size, and brightness by comparing themeasured light-intensity values with total radiance values generated asa function of depth of a point-source light emitters, wherein the totalradiance values are generated from a model of photon-diffusion from alight source of defined size, shape, and/or depth below a body surface.8. The method of claim 1, wherein said refining includes using dataobtained from a computer simulation of the diffusion of light from alight-emitting source in a turbid medium.
 9. The method of claim 8,wherein said computer simulation is a photon diffusion model.
 10. Themethod of claim 9, wherein said refining comprises (i) generating aplurality of theoretical light-intensity profiles, based on a model ofphoton diffusion from a light-emitting source located at one of aplurality of depths, and having one of a plurality of sizes and shapes,through a turbid medium having absorption and scattering propertiessimilar to those of biological tissue, (ii) comparing the quality of fitbetween each of said plurality of theoretical light-intensity profilesand said first measured light intensity profile, (iii) selecting thetheoretical light intensity profile which provides to the first measuredlight intensity profile, and (iv) obtaining an approximate depth, shapeand brightness of the source in the subject using parameters from thetheoretical light intensity profile selected in (iii).
 11. The method ofclaim 10, wherein comparing the quality of fit is done using a leastsquares algorithm.
 12. The method of claim 10, wherein comparing thequality of fit is done using genetic algorithms.
 13. The method of claim10, wherein said generating includes employing in a photon-scatteringmodel, one or more predetermined tissue-specific light-scatteringcoefficients corresponding to tissue through which said photons travel.14. The method of claim 1, wherein said light-emitting source is aluminescent moiety.
 15. The method of claim 1, wherein saidlight-emitting source is a fluorescent moiety.
 16. The method of claim1, wherein said light-emitting source is administered to the subject andbinds to a selected target in the subject prior to said measuring. 17.The method of claim 1, wherein said light-emitting source is alight-generating protein.
 18. The method of claim 17, wherein saidlight-generating protein is expressed by biological cells of saidsubject.
 19. The method of claim 17, wherein said light-generatingprotein is expressed by biological cells administered to said subject.20. The method of claim 1, further comprising generating a visualrepresentation of said light-emitting source, using the approximatedepth and shape of the source in the subject, and superimposing saidvisual representation onto an image of the subject.
 21. The method ofclaim 20, wherein said image is a two-dimensional image.
 22. The methodof claim 20, wherein said image is a three-dimensional image. 23.Apparatus for use in investigating the location and size of alight-emitting source in a subject, comprising: a light-tight enclosurewithin which light-emission events from the subject can be detected, anoptical system contained within the enclosure for use in obtaining afirst light intensity profile constructed by measuring, from a firstperspective within the enclosure, photons which (i) originate from thelight-emitting source, (ii) travel through turbid biological tissue ofthe subject, and (iii) are emitted from a first surface region ofinterest of the subject; and a computational unit operatively connectedto the photo-detector for (i) fitting said first measured lightintensity profile with a parameter-based biophotonic function; and (ii)refining the parameters of the biophotonic function, using data otherthan said first measured light intensity profile, to generate anapproximate depth, shape, and brightness of the source in the subject.24. The apparatus of claim 23, wherein the optical system includes acharged-coupled-device (CCD) that can be operated in a cooled condition,and a lens for focusing light onto the CCD.
 25. The apparatus of claim24, wherein the optical system is designed to detect photons emittedfrom a plurality of different selected surface regions of interest ofthe subject.
 26. The apparatus of claim 23, wherein the optical systemincludes one or more wavelength filters for transmitting photons withindifferent selected wavelength ranges, for use in measuringphoton-emission intensities within two or more different selectedwavelength ranges between about 400-800 nm, and the computation unit isoperable to carry out at least one of the parameter-refinementoperations: (i) determining the relative light intensities measured atthe different wavelengths, and comparing the determined relative lightintensities with known relative signal intensities at said differentwavelengths, as a function of tissue depth; and (ii) comparing themeasured spectrum with a plurality of spectra measured fromlight-emitting sources placed at various depths within tissue, anddetermining the depth of the light-emitting target region from matchingthe measured spectrum with the known spectra.
 27. The apparatus of claim23, wherein said computational unit includes a data file containingpredetermined spatial distribution information relating the spatialdistribution of light emitted within the selected wavelength ranges as afunction of size, shape, and/or depth of the a model target's lightemission within a model body region of a model subject, and the unitfunctions in carrying out task (i) to compare spectral characteristicsof the first light-intensity profile with those contained in thedatabase.
 28. The apparatus of claim 23, wherein the computational unitis operable to integrate the intensity pattern image into a singleintensity value, and to estimate source size by integrated lightintensity values generated as a function of a light-emitting source of aparticular size and shape.
 29. The apparatus of claim 23, wherein saidcomputational unit includes a database containing a plurality oftheoretical light-intensity profiles, based on a model of photondiffusion from a glowing source located at one of a plurality of depths,and having one of a plurality of sizes and shapes, through a turbidmedium having absorption and scattering properties similar to those ofbiological tissue, and the unit is operable to (i) compare the qualityof fit between each of said plurality of theoretical light-intensityprofiles and first measured light intensity profile, (ii) select thetheoretical light intensity profile which provides the best fit to thefirst measured light intensity profile, and (iii) obtain an approximatedepth and shape of the source in the subject using parameters from thetheoretical light intensity profile selected in (ii).
 30. The apparatusof claim 23, wherein the computational unit is operable to generate avisual 2-, or 3-dimensional representation of said light-emittingsource, using the approximate depth and shape of the source in thesubject, and superimposing said visual representation onto a 2- or3-dimensional image of the subject.
 31. The method of claim 1, whereinsaid obtaining includes obtaining absolute intensity values.
 32. Amethod of determining the depth of a light-emitting source in tissue ofa subject, comprising: (a) measuring light emission intensity from thesubject at two or more different wavelengths between about 400 and about1000 nm; (b) obtaining information related to optical properties ofsubject's tissue, and (c) using the measured light intensities and theobtained information to determine the depth of the light-emittingsource.
 33. The method of claim 32, wherein step (a) is carried out bymeasuring light intensities at each at two different wavelengths, step(b)includes measuring the coefficients of attenuation and diffusion intissue or material corresponding to that of the subject, and step (c)includes using said coefficients in a formula to estimate depth of saidsource.
 34. The method of claim 33, wherein step (b) is carried out bydetermining, at two or more different wavelengths, light intensitiesfrom by a light-emitting source located at each of a plurality of depthsin tissue or material corresponding to that of the subject, and step (c)is carried out by matching the measured light intensities with referencelight intensities determined at each of the plurality of depths.
 35. Themethod of claim 34, wherein step (a) includes measuring a spectralprofile of light intensities from the light-emitting source, step (b) iscarried out by determining spectral profiles of light intensities from alight-emitting source at each of a plurality of depths, and step (c) iscarried out by matching the measured profile with the determinedprofiles, to identify a best curve fit.